title: Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay.
abstract: Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking. The aim of this study was to evaluate the most practical and routinely implementable published PCR assays designed for C. fetus species and subspecies identification. The sensitivity and specificity of the assays were calculated by using an extensively characterized and diverse collection of C. fetus strains. AFLP and MLST identification were used as reference. Two PCR assays were able to identify C. fetus strains correctly at species level. The C. fetus species identification target, gene nahE, of one PCR assay was used to develop a real-time PCR assay with 100% sensitivity and 100% specificity, but the development of a subspecies venerealis specific real-time PCR (ISCfe1) failed due to sequence variation of the target insertion sequence and prevalence in other Campylobacter species. None of the published PCR assays was able to identify C. fetus strains correctly at subspecies level. Molecular analysis by AFLP or MLST is still recommended to identify C. fetus isolates at subspecies level.
authors.affiliation: Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
authors.forename: Linda
authors.initials: L
authors.lastname: van der Graaf-van Bloois
authors.forename: Marcel A P
authors.initials: MA
authors.lastname: van Bergen
authors.forename: Fimme J
authors.initials: FJ
authors.lastname: van der Wal
authors.forename: Albert G
authors.initials: AG
authors.lastname: de Boer
authors.forename: Birgitta
authors.initials: B
authors.lastname: Duim
authors.forename: Tracy
authors.initials: T
authors.lastname: Schmidt
authors.forename: Jaap A
authors.initials: JA
authors.lastname: Wagenaar
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D002169:Campylobacter Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D002168:Campylobacter fetus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000382:microbiology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: AFLP; Bovine genital campylobacteriosis; Campylobacter fetus; MLST; Real-time PCR; Subspecies identification